Prenatal diagnosis or prenatal screening refers to testing for diseases or conditions in a fetus or embryo before it is born. The aim is to detect birth defects such as neural tube defects, Down syndrome, chromosome abnormalities, genetic diseases and other conditions, such as spina bifida, cleft palate, Tay Sachs disease, sickle cell anemia, thalassemia, cystic fibrosis, Muscular dystrophy, and fragile X syndrome. Screening can also be used for prenatal sex discernment. Common testing procedures include amniocentesis, ultrasonography including nuchal translucency ultrasound, serum marker testing, or genetic screening. In some cases, the tests are administered to diagnose high-risk pregnancies early so that delivery can be scheduled in a tertiary care hospital where the baby can receive appropriate care.
Diagnostic prenatal testing can be by invasive or non-invasive methods. An invasive method involves probes or needles being inserted into the uterus, e.g. amniocentesis, which can be done from about 14 weeks gestation, and usually up to about 20 weeks, and chorionic villus sampling, which can be done earlier (between 9.5 and 12.5 weeks gestation, but which may be slightly more risky to the fetus. Chorionic villi sample and amniocentesis have related miscarriage risks of approximately 1 in 100 pregnancies and 1 in 200 pregnancies, respectively. Less risky procedures for non-invasive prenatal diagnosis have been implemented in the US and other countries. These techniques include examinations of the woman's womb through ultrasonography and maternal serum screens. For example, blood tests for select trisomies based on detecting fetal DNA present in maternal blood have become available (e.g., tests for Down syndrome in the United States and tests for Down and Edwards syndromes in China). The presence of fetal DNA in maternal plasma was first reported in 1997, offering the possibility for non-invasive prenatal diagnosis simply through the analysis of a maternal blood sample (Lo et al (1997), Lancet 350:485-487).
As technology progresses, tests will shift from current more risky tests to less risky non-invasive tests. Leading medical bodies (e.g., the American College of Obstetricians and Gynecologists, the American College of Medical Genetics and Genomics, and the Society of Maternal and Fetal Medicine) currently endorse non-invasive prenatal screening for high-risk pregnancies. In addition, several companies (e.g., Sequenom, Verinata, Ariosa, Natera) are offering aneuploid testing services (e.g., to detect trisomy of chromosomes 21, 18, 13, X, and Y) based on laboratory-developed tests (LDT) developed under the Clinical Laboratory Improvement Amendments (CLIA) program. Furthermore, several payors (e.g. BCBS, Kaiser) offer reimbursement for trisomy testing in the face of increasing consumer demand driven by the overwhelming desire by expectant mothers to opt for modern non-invasive testing alternatives.
One particularly advantageous non-invasive test involves the analysis of cell-free fetal DNA (cffDNA). In a particular application, non-invasive prenatal aneuploidy testing of cffDNA is predicated on detecting the small fractional excess of DNA exhibited in instances of aneuploidy trisomy) compared to a normal euploid fetus. In these tests, trisomy detection represents a problem of distinguishing 3 copies from 2 copies of a chromosome in a mixture where approximately 90% of the sample is euploid (e.g., disomic).
However, in practice, circulating cffDNA constitutes a minor fraction (approximately 3% to 6% (see, e.g., Lo et al. (1998) Am J Hum Genet 62: 768) or up to 10% to 20% according to some measures (see, e.g., Lun et al (2008) Clin Chem 64: 1664) of the total cell-free DNA in maternal plasma. In general, fractional circulating cffDNA concentration averages approximately 10% in early pregnancy (see, e.g., Chiu et al (2011) BMJ 342: c7401). This limitation poses a considerable challenge for non-invasive prenatal testing strategies that rely on direct chromosome enumeration methods for detecting fetal aneuploidy status (such as digital PCR, next-generation sequencing, or mass spectrometry).
For instance, assuming a 10% fetal DNA content in maternal plasma, the fractional increase of DNA in a fetal trisomy (e.g., involving chromosome 13, 18, 21, X, Y, or another chromosome) compared to a normal fetus is expected to be 1.05 (that is, 21 total copies for a trisomy compared to 20 copies for euploidy). This subtle difference in DNA content is measured by ultra-high density statistical counting methods that discriminate between the 1 and 1.05 ratio values observed in normal euploid and trisomic pregnancy cases, respectively.
In the routine clinical setting, the fetal DNA content of maternal plasma is commonly less than 10%, resulting in even smaller chromosomal disparities between trisomies and euploidies (e.g., ratios of approximately 1.02 to 1.03). The ability to enrich the cffDNA fraction by several-fold to modest levels (e.g., approximately 5-fold to 10-fold enrichment resulting in approximately 25% to 40% fetal DNA content) reduces the coverage and/or partition requirements for NGS and digital PCR applications, respectively (e.g., decreases the “digital real estate” associated with the technologies). Fetal DNA enrichment also facilitates fetal aneuploidy detection by mass spectrometry-based methods. Consequently, technologies are needed to enrich maternal blood samples for cffDNA to improve prenatal non-invasive diagnostic testing.